Project Summary/Abstract - Project 4: Mechanistic Basics of FLASH Effect: Role of O2 Recently, exciting and unexpected new data in several animal models strongly supports the premise that ultra- high dose rate irradiation termed FLASH radiotherapy (FLASH-RT) can result in significant sparing of normal tissue while preserving tumor responses, effectively leading to significant increases in the therapeutic window for improving radiotherapy outcomes. However, the mechanisms for this effect are currently unknown. Project 4 will build on the exciting preliminary data that carbogen (95% O2; 5% CO2) breathing significantly diminished the protection of normal brain tissue while enhancing the responses of tumors. This result as well as theoretical considerations of the differential redox reactions of O2 in cancer versus normal tissues provides the impetus to study the reactions of O2 as drivers of the differential responses of normal and cancer tissue to FLASH-RT. These considerations have led to the central hypothesis in Project 4 that differential metabolism of organic hydroperoxides (ROOH) in cancer versus normal tissues following FLASH-RT, caused by differences in labile Fe pools and lipid peroxidation chain reactions, mediates the differential responses of tumor vs. normal tissues to FLASH-RT. This hypothesis will be pursed in the following Aims: Specific Aim 1: Determine how varying the O2 tension that animals breathe alters the structural integrity of the vasculature, mature neuronal morphology, activation of inflammatory responses, and neurocognitive effects of FLASH-RT versus conventional dose rate exposure. Specific Aim 2: Determine if changes in O2 in tumor versus normal tissues following FLASH-RT using Oxylite measurements correlate with measurements of ROOH and downstream products of lipid peroxidation in animal tissues exposed to FLASH-RT at doses where normal tissue sparing is observed. Specific Aim 3: Determine the causal involvement of organic hydroperoxides and redox active metal ions in the differential sensitivity of tumor versus normal tissues using pharmacological and genetic approaches to manipulate glutathione peroxidases (GPx) 1 and 4 as well as metal chelators that inhibit Fe redox cycling. Scientific Impact: The successful completion of this project will clearly define biochemical mechanisms involving organic hydroperoxide metabolism and redox active iron metabolism underlying the selective sparing of normal tissues from FLASH-RT in mouse models of brain cancer therapy as well as providing a new paradigm for using FLASH-RT to exploit fundamental differences in cancer vs. normal cell metabolism for increasing treatment efficacy while protecting normal tissues. Integration into the P01: Project 4 will interact with all projects in the P01 by providing genetically manipulated brain cancer cells that conditionally overexpress GPx 1 and 4 as well as ferritin and validated biomarkers of oxidative stress for testing mechanisms of oxidative metabolism in models of FLASH-RT.